Abstract:
MyD88 and IRAK-4 are two intracellular proteins that are involved in innate immunity. They form an oligomeric complex known as the Myddosome when a Toll-like receptor is activated upon ligand binding. This complex forms part of post-receptor signalling and is assembled by the interaction of death domains of both proteins. The regulatory mechanisms of assembly and dissociation of the Myddosome are not yet fully understood. One possible mechanism is that autophosphorylation regulates the assembly of the Myddosome.
IRAK-4 is a serine/threonine kinase that phosphorylates another kinase, IRAK-1 during TLR signaling. In addition to phosphorylating IRAK-1, it also phosphorylates itself, a process known as autophosphorylation. Its autophosphorylation is an intramolecular event that starts in the kinase loop and extends to its death domain. It is reported that two autophosphorylation sites namely serine 8 and threonine 62 were mapped to its death domain. We substituted these two amino acids with aspartic acid to create phosphomimetics. The mutant phosphomimetics were expressed in E. coli cells and thereafter purified with the aim of determining if they will form the Myddosome when mixed with MyD88 death domain.
Wild type and phosphomimetic complexes were formed and characterized using size exclusion chromatography, dynamic scattering and analytical ultracentrifugation. Our results showed that phosphomimetic mutants were able to form an oligomeric complex similar in size to the Myddosome. This result was contrary to what has been reported in literature. We therefore concluded that phosphomimetics were not ideal replacements for phosphorylation.
